Depicting Fecal Microbiota Characteristic in Yak, Cattle, Yak-Cattle Hybrid and Tibetan Sheep in Different Eco-Regions of Qinghai-Tibetan Plateau

ABSTRACT The gut microbiota is closely associated with the health and production performance of livestock. Partial studies on ruminant microbiota are already in progress in the Qinghai-Tibetan Plateau Area (QTPA) in China, but large-scale and representative profiles for the QTPA are still lacking. Here, 16S rRNA sequencing was used to analyze 340 samples from yak, cattle, yak-cattle hybrids, and Tibetan sheep, which lived in a shared environment from 4 eco-regions of the QTPA during the same season, and aimed to investigate the fecal microbiota community composition, diversity, and potential function. All samples were clustered into 2 enterotypes, which were derived from the genera Ruminococcaceae UCG-005 and Acinetobacter, respectively. Environment, human activity, species, and parasitization all affected the fecal microbiota. By assessing the relationship between the fecal microbiota and the above variables, we identified a scattered pattern of fecal microbiota dissimilarity based more significantly on diet over other factors. Additionally, gastrointestinal nematode infection could reduce the capacity of the bacterial community for biosynthesis of other secondary metabolites, carbohydrate metabolism, and nucleotide metabolism. Ultimately, this study provided a fecal microbiota profile for ruminants living in 4 eco-regions of the QTPA and its potential future applications in developing animal husbandry regimes. IMPORTANCE Cattle, yak, and sheep reside as the main ruminants distributed throughout most regions of Qinghai-Tibetan Plateau Area (QTPA) in China. However, there is a lack of large-scale research in the QTPA on their fecal microbiota, which can regulate and reflect host health as an internalized “microbial organ.” Our study depicted the fecal microbiota community composition and diversity of yak, cattle, yak-cattle hybrids, and Tibetan sheep from 4 eco-regions of the QTPA. Additionally, our results demonstrated here that the ruminant samples could be clustered into 2 enterotypes and that diet outweighed other factors in shaping fecal microbiota in the QTPA. This study provided a basis for understanding the microbiota characteristic of ruminants and its possible applications for livestock production in the QTPA.

16S rRNA sequencing to analyze 340 samples from yak, cattle, yak-cattle hybrid and Tibetan sheep living in four regions of Qinghai-Tibetan Plateau.

Specific comments:
Introduction: Lines 60-69: The context of the study is described but the problem is not. What is the problem that justifies analyzing the fecal microbiota of four ruminants? Is it the presence of parasites, is it based on geographical differences? Without a clearly stated hypothesis or research question, this study is only descriptive although the number of fecal samples is high. Please identify the problem.
Lines 70-71: Why did you also study the fecal microbiota of Tibetian sheep? Besides being a ruminant, what is the common problem with cattle, yak, yak-cattle hybrids that justifies including them in the study?
Discussion: Lines 258-281: Enterotyping was initially proposed to differentiate gut microbiota profiles in order to associate them with predispositions or their diet for human. In this study, enterotyping in ruminants appears to be only a possible environmentrelated grouping. Why is the presence of two enterotypes of interest to the advancement of knowledge for ruminants ? This point does not seem to have been clearly specified?
Reviewer #2 (Comments for the Author):

General comments
The authors investigated an interesting studying on the fecal microbiota profiles of domestic ruminants (cattle, yak, yak-cattle hybrid, and Tibetan sheep) dwelling in 4 typical eco-regions with homologous longitudes in Qinghai-Tibetan Plateau Area.
However, the overall quality is relatively weak, especially for the experimental design. There are only 340 samples in this study, but with too many interference factors, such as the sex, age, environment, feed, infections, animal breed, etc.
In addition, the objective of this study is not very clear. If you aim to detect the effect of environment on the gut microbiota in ruminants. The animals and feed should be similar. And what is the meaning or implication of this study that has not been clearly clarified?
There are many grammar and format issues that should be corrected throughout the manuscript. For instance: Line-by-line comments Line 23-24: suggest changing to "All of the environment, human activity, species, and parasitization affected the fecal microbiota." Line 65: suggest changing to "taken into; a big natural; and similar biological" Line 349-352: suggest using parenthesis rather than square brackets Reference: Please pay more attention to the format of the Reference following the instruction to authors. Such as 19, 40, 41, 46, ...

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Dear Editor and Reviewer,
We would like express our gratitude to the Editor and Reviewer for the earnest and instructive comments and suggestions which would help us to improve the quality of the manuscript. Additionally, the text added in the revised manuscript was highlighted in yellow. The modification and response as follow.

Response to Reviewer 1 Comments
Lines 60-69: The context of the study is described but the problem is not. What is the problem that justifies analyzing the fecal microbiota of four ruminants? Is it the presence of parasites, is it based on geographical differences? Without a clearly stated hypothesis or research question, this study is only descriptive although the number of fecal samples is high. Please identify the problem. RESPONSE: Thank you very much for your suggestion. Sorry for not communicating clearly. The prime objective in this study is to depict the profile of fecal microbiota of ruminants in the QTPA, and then evaluating the dominant factor that shift microbiota structure, among several factors such as diet (environment), genetics and parasitization. This part has been arranged to suit your suggestion.
Lines 70-71: Why did you also study the fecal microbiota of Tibetian sheep? Besides being a ruminant, what is the common problem with cattle, yak, yak-cattle hybrids that justifies including them in the study? RESPONSE: Thank you very much for pointing it out. As the manuscript initially outlined that cattle, yak and sheep were the main ruminants in the Qinghai-Tibetan Plateau Area (QTPA). We try analyse the fecal microbiota of these main ruminants to reveal the fecal microbiota characteristic of ruminants in the QTPA. Moreover, the results showed similar conclusions about the importance of environment factor on shifting the fecal microbiota either in sheep or in cattle, yak and yak-cattle hybrids.
Lines 258-281: Enterotyping was initially proposed to differentiate gut microbiota profiles in order to associate them with predispositions or their diet for human. In this study, enterotyping in ruminants appears to be only a possible environment-related grouping. Why is the presence of two enterotypes of interest to the advancement of knowledge for ruminants? This point does not seem to have been clearly specified? RESPONSE: Thanks very much for your advice. High-altitude animals have evolved physiological adaptations to live in extremely conditions of the QTPA, and for animals, enterotype is an instinctive response to environment. This study not only helped determine the enterotype for ruminants in the QTPA, but also reflected the information of the local vegetation and ecology. Related content has been added to line 281-283 (clean revision).

Response to Reviewer 2 Comments
However, the overall quality is relatively weak, especially for the experimental design. There are only 340 samples in this study, but with too many interference factors, such as the sex, age, environment, feed, infections, animal breed, etc. RESPONSE: Thank you very much for your suggestion. By agreeing that the sex, age, environment, feed, infections, animal breed could affect the fecal microbiota. Here, even with variables (the sex and age), results still showed the environmental tendency of the fecal microbiota, environment over other factors in shaping the microbiota structure. Furthermore, in this study, sampling, preservation condition, experimental methods and sequencing of all samples were carried out under the same conditions, to reduce the error. As mentioned at the end of the manuscript, this study demonstrated the importance of the environment, but there are still many more samples required to explore and perfect the spectrum of fecal microbiota of ruminants in the QTPA.
In addition, the objective of this study is not very clear. If you aim to detect the effect of environment on the gut microbiota in ruminants. The animals and feed should be similar. And what is the meaning or implication of this study that has not been clearly clarified? RESPONSE: Thanks very much for your suggestion. Sorry for not communicating clearly. Due to diet is part of environment factors, and diet is acquiesced as environment factor here. The effect of environment (diet) was demonstrated from varied ecotypes in different typical eco-regions, such as yaks from 4 eco-regions and Tibetan sheep from 3 eco-regions, in which shared the same breeds but diverse regions. The genetics or species influence was confirmed by multiple species or varieties (cattle, yak and yak-cattle hybrid) in one habitat (Diqing), which grew under similar environment and forage.
There are many grammar and format issues that should be corrected throughout the manuscript. For instance: Fig. or FIG; Amplicon Sequence Variants or amplicon sequence variants; Studies on ruminant microbiota has been already should be "Studies on ruminant microbiota have been..." RESPONSE: Thanks so much for your reminding. the above questions have been modified and the grammar has been checked throughout the whole manuscript.
Line-by-line comments Line 23-24: suggest changing to "All of the environment, human activity, species, and parasitization affected the fecal microbiota." Line 65: suggest changing to "taken into; a big natural; and similar biological" Line 349-352: suggest using parenthesis rather than square brackets RESPONSE: Thank you very much. We have modified the above questions as you suggested. Reference: Please pay more attention to the format of the Reference following the instruction to authors. Such as 19, 40, 41, 46, ... RESPONSE: Thanks so much for alerting us to the problem. The format has been fixed. Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Reviewers' comments: 1) However, the overall quality is relatively weak, especially for the experimental design. There are only 340 samples in this study, but with too many interference factors, such as the sex, age, environment, feed, infections, animal breed, etc. RESPONSE: Thank you very much for your suggestion. By agreeing that the sex, age, environment, feed, infections, animal breed could affect the fecal microbiota. Here, even with variables (the sex and age), results still showed the environmental tendency of the fecal microbiota, environment over other factors in shaping the microbiota structure. Furthermore, in this study, sampling, preservation condition, experimental methods and sequencing of all samples were carried out under the same conditions, to reduce the error. As mentioned at the end of the manuscript, this study demonstrated the importance of the environment, but there are still many more samples required to explore and perfect the spectrum of fecal microbiota of ruminants in the QTPA. New Comments: I still suggest that the authors should list out the sex and age, and show their effects in different region.
2) In addition, the objective of this study is not very clear. If you aim to detect the effect of environment on the gut microbiota in ruminants. The animals and feed should be similar. And what is the meaning or implication of this study that has not been clearly clarified? RESPONSE: Thanks very much for your suggestion. Sorry for not communicating clearly. Due to diet is part of environment factors, and diet is acquiesced as environment factor here. The effect of environment (diet) was demonstrated from varied ecotypes in different typical eco-regions, such as yaks from 4 eco-regions and Tibetan sheep from 3 eco-regions, in which shared the same breeds but diverse regions. The genetics or species influence was confirmed by multiple species or varieties (cattle, yak and yak-cattle hybrid) in one habitat (Diqing), which grew under similar environment and forage. New Comment: We know that the diet is included into the environment factors, but you can not say that diet is acquiesced as environment. Thus, in the conclusion you mention that the fecal microbiota dissimilarity based on environment rather than host factor among ruminants in the QTPA. The environment should be the most significant factors. What is new from your study? If your objective is to show the difference of fecal microbiota among the four regions, you need list out them in the conclusion, rather than just say the environment rather than host factor. Please make the aim of this study consistent and more clear.

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